Project Summary Many aspects of cell biology as well as tissue physiology and the proper functioning of organisms are essentially problems in material science. The structures and reactions that enable proper functioning of an organism need to produce movements that are greater than those generated by random Brownian motion. Cells need to build structures that are strong enough to resist gravitational forces as well as the mechanical stresses that are generated by the same molecular structures and cellular assemblies that evolved to generate movement and force. A related problem in soft matter is to understand the physical chemistry and dynamics of the phospholipid bilayer that forms the cell membrane and orchestrates the signals generated at the cell membrane and sent to the interior. This MIRA application combines two physical studies. One is focused on the mechanical properties of purified biopolymer networks, intact cells, and whole tissues. The second involves biophysical and biochemical characterizations of lipid bilayers containing anionic signaling lipids to determine how these lipids distribute in the dynamic membrane and how this organization impacts their control of intracellular protein targets. We have characterized and worked with theorists to explain the striking nonlinear elastic response of semi-flexible polymeric networks, with emphasis on the cytoskeletal intermediate filament protein vimentin, and shown how these physical models help explain cell and tissue mechanism. We have also shown how important viscoelastic properties of the substrate are to cell phenotypes and have developed new materials by which to study them. In membrane studies, we collaborate with molecular dynamics experts to produce a coherent model of the structures and motions of anionic signaling lipids such as PIP2 ranging from the atomic to the molecular, to the macroscopic membrane scale. Biochemical and cellular studies show that the spatial distribution of these lipids in bilayers impacts the way they control cytoskeletal actin assembly at the cytoplasm/membrane interface. Future work will build on these studies in three different areas. We will use our established models of semiflexible networks to determine why vimentin networks, in contrast to those formed by stiffer polymers, become stiffer when compressed, whereas crosslinked actin or microtubules become softer. We will also extend our studies of extracellular polymers and cells to intracellular systems: cytoskeletal networks containing membrane-bounded organelles, and crosslinked DNA or chromatin with the liquid particles and organelles contained in the nuclear matrix. Here we will use our newly developed method to prepare intact metabolically active nuclei surrounded by a thin layer or cytoplasm and a plasma membrane, and determine how the perinuclear vimentin cage influences the structure and mechanical response of the nucleus. Membrane studies will use our previous methods to alter PIP2 distribution in artificial bilayers and isolated cell membranes, to study how similar changes in PIP2 distribution triggered by changes in intracellular Ca2+ or cholesterol affect actin assembly in intact cells. We will also build on the MD simulations of relatively small membrane systems to coarse grain simulations using the essential features identified by current all-atom simulations. These will enable studies of systems that are large enough and followed for sufficient time to produce phase transitions and nano-scale lipid clusters. These models will be used to predict how different PIP2 binding proteins respond to lateral distribution of the lipid and test these ideas biochemically and in cells.